S139: BCOR DELETION SUSTAINS NOTCH1 SIGNALLING ACTIVATION TO ACCELERATE CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) PROGRESSION TOWARD RICHTER TRANSFORMATION IN MICE
Authors: C. Rompietti, D. Sorcini, F. De Falco, E. Dorillo, F. M. Adamo, E. C. Silva Barcelos, A. Stella, A. Scialdone, A. Esposito, R. Arcaleni, B. Bigerna, G. Martino, L. Moretti, M. G. Mameli, C. Geraci, L. Sandoletti, A. Cipiciani, E. Rosati, B. Falini, P. Sportoletti
Journal: HemaSphere (2022)

Background: BCL6 co-repressor (BCOR) is a transcription factor involved in various biological processes including lymphoid development. BCOR disruptive mutations were found in up to 2% of CLL and frequently associated with aberrations of NOTCH1, one of the most common genetic alterations with poor prognosis in CLL and Richter transformation (RT). Recent evidence also indicates that BCOR is involved in NOTCH signalling suppression during embryogenesis. These data indicate the need for further investigation on the role of BCOR mutation and its interplay with NOTCH1 in CLL pathogenesis. Aims: We aim to elucidate the impact of Bcor deficiency in CLL and RT, focusing on the role of active NOTCH1 signalling. Methods: We used a conditional knockout mouse of Bcor (Sportoletti et al, Leukemia 2021) crossed with CD19-Cre mice, to specifically delete Bcor in B-cells, and with the Eμ-TCL1 mouse model of CLL. Mice were characterized for disease phenotype (using flow-cytometry and histological analyses), overall survival and drug response. NOTCH1 activity was assessed by western blot (WB) for the NOTCH1-intracellular domain (NOTCH1-IC) and HES1 expression. Results: B-cell restricted loss of Bcor in Eµ-TCL1 mice significantly expanded leukemic CD5+CD19+ cells in the peripheral blood (PB; p<0.05), spleen (p<0.01), bone marrow (BM; p<0.001), compared to Eµ-TCL1 control mice. No leukemic cells were found in BcorCD19Cre+ mice. Cellular changes resulted in a poorer survival of double mutant mice compared to single-mutant and wild-type (WT) controls (median survival of 334 days vs unreached in BcorCD19Cre+;Eμ-TCL1 vs the other groups, respectively). Adoptive transfer of spleen cells (CD5+CD19+ leukemic burden >50%) resulted in a more rapidly lethal disease in recipient of BcorCD19Cre+;Eμ-TCL1 compared to Eμ-TCL1 cells (median survival of 37.5 vs 74 days, respectively, p<0.05; Figure1A). At necropsy, double mutant mice presented massive splenomegaly, whose histopathological examination revealed a diffuse infiltration by high-mitotic blastoid cells, significantly increased in size, defining a high-grade lymphoid malignancy distinct from the leukemia of Eμ-TCL1 counterparts (Fig.1B). In order to gain mechanistic insight relating to the phenotypic observations, we measured NOTCH1 activity. BcorCD19Cre+;Eμ-TCL1 splenic CD5+CD19+ cells showed significantly increased levels of the active NOTCH1-IC and the up-regulation of its direct target HES1, compared to leukemic cells from Eμ-TCL1 mice and CD19+ cells from non-leukemic BcorCD19Cre+ and WT mice used as control (Fig.1C). In vivo treatment with the NOTCH1 inhibitor Bepridil resulted in a significant growth delay of CD5+CD19+ cells in the PB of mice transplanted with BcorCD19Cre+;Eμ-TCL1 leukemic cells compared to vehicle. At sacrifice, Bepridil caused a significant reduction of spleen dimensions and CD5+CD19+ cellularity, associated with reduced levels of active NOTCH1-IC, c-MYC and HES1 compared to vehicle (28%, 54% and 38% reduction, respectively). NOTCH1 inhibition significantly improved the survival of diseased mice (median survival of 39 vs 33 days in Bepridil vs vehicle, respectively; N=4, p<0.05). Image:Summary/Conclusion: We showed for the first time the tumour suppressor activity of Bcor in a CLL mouse model, ultimately leading to transformation towards a high-grade lymphoma, mimicking human RT. Mechanistically, we implied NOTCH1 signalling activation in Bcor loss mediated tumorigenesis with potential for targeted treatment for high-risk CLL and RT patients.